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PubMed1985

Chemotherapeutic attack of hypoxic tumor cells by the bioreductive alkylating agent mitomycin C.

Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiromycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent. Instead, DT-diaphorase appears to metabolize mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid tumors. Using X-rays to eliminate well oxygenated cells of a solid tumor implant of the EMT6 carcinoma, we have found that the combination of dicoumarol plus mitomycin C is more toxic to hypoxic tumor cells in vivo than mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of mitomycin C activation permits a prediction of which tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.

Animals↗

PubMed2004

Intravesical bacillus Calmette-Guérin is superior to mitomycin C in reducing tumour recurrence in high-risk superficial bladder cancer: a meta-analysis of randomized trials.

OBJECTIVE: To assess, in a systematic review and meta-analysis, the relative effectiveness of intravesical mitomycin C and bacillus Calmette-Guérin (BCG) for tumour recurrence, disease progression and overall survival in patients with medium- to high-risk Ta and T1 bladder cancer. METHODS: The major medical databases were searched comprehensively up to June 2003, and relevant journals hand-searched for randomized controlled trials, in any language, that compared intravesical mitomycin C with BCG in medium- to high-risk patients with Ta or T1 bladder cancer. RESULTS: Twenty-five articles were identified but only seven were considered eligible for the analysis. This represented 1901 evaluable patients in all, 820 randomized to mitomycin C and 1081 to BCG. Six trials had sufficient data for meta-analysis and included 1527 patients, 693 in the mitomycin and 834 in the BCG arm. There was no significant difference between mitomycin C and BCG for tumour recurrence in the six trials, with a weighted mean log hazard ratio, LHR, (variance) of -0.022 (0.005). However, there was significant heterogeneity between trials (P = 0.001). A subgroup analysis of three trials that included only high-risk Ta and T1 patients indicated no heterogeneity (P = 0.25) and a LHR for recurrence of -0.371 (0.012). With mitomycin C used as the control in the meta-analysis, a negative ratio is in favour of BCG and, in this case, was highly significant (P < 0.001). The seventh trial (in abstract form only) used BCG in low doses for two arms of the trial (27 mg and 13.5 mg) compared with a standard dose of mitomycin C (30 mg), and reported a significantly lower recurrence rate with BCG (27 mg) than for mitomycin C (P = 0.001). Only two trials included sufficient data to analyse disease progression and survival, representing 681 patients (338 randomized to BCG and 343 to mitomycin C). There was no significant difference between mitomycin C and BCG for disease progression, with a LHR of 0.044 (0.04) (P = 0.16), or survival, at -0.112 (0.03) (P = 0.50). Adverse events were slightly more frequent with BCG. Local toxicity (dysuria, cystitis, frequency and haematuria) were associated with both mitomycin C (30%) and BCG (44%). Systemic toxicity, e.g. chills, fever and malaise, occurred with both agents (12% and 19%, respectively) although skin rash was more common with mitomycin C. CONCLUSION: Tumour recurrence was significantly lower with intravesical BCG than with mitomycin C only in those patients at high risk of tumour recurrence. However, there was no difference in disease progression or survival, and the decision to use either agent might be based on adverse events and cost.

Adjuvants, Immunologic↗

PubMed1999

Quantification of intraoperative administration of mitomycin-C in filtering surgery with surgical sponge material.

PURPOSE: To determine the absorption and release of mitomycin-C 0.4 and 0.2 mg/mL from sponge-like specimens of Spongostan film (Ferrosan, Copenhagen, Denmark) and the scleral and conjunctival impregnation in an experimental model of filtering surgery. METHODS: The maximum amount of mitomycin per volume unit that Spongostan is able to absorb was determined physically as the difference between dry weight and soaked weight. Mitomycin-C activity in known volumes of Spongostan after mitomycin-C release in vitro also was determined at 0, 1, 10, and 30 seconds and 1, 3, 5, 10, 15, and 30 minutes. Antibiotic activity of the specimens was evaluated by means of bioassay. Millimeters of inhibition of bacterial growth were related to microg of mitomycin activity according to a reference curve obtained from known amounts of mitomycin-C. Finally, 10 eyes of 10 rabbits underwent filtering surgery with intraoperative application of mitomycin by means of the Spongostan film. The Spongostan implants then were removed and tested for mitomycin activity. Scleral and conjunctival specimens were obtained for bioassay. RESULTS: The maximum capacity of 25 mm2 x 0.5 mm thick Spongostan films saturated in 0.4 and 0.2 mg/mL solutions of mitomycin-C were 8.49 microg and 4.23 microg, respectively. Biologic activity (bioassay determination) was 8.24 microg and 4.19 microg of mitomycin-C, respectively. In vitro release of mitomycin was gradual until 30 minutes. In vivo mitomycin release from Spongostan after 5 minutes was 6.91 microg. Impregnation with the antimitotic was better in conjunctiva than sclera. CONCLUSION: Bioassay permits quantification of mitomycin-C activity. The release from sponge specimens is gradual, and impregnation was better in conjunctiva than sclera.

Absorption↗

PubMed1987

Presence of mitomycin resistant T cells in peripheral blood of normal individuals.

T cell colonies can be easily grown from peripheral blood, and are an index of cellular immunocompetence. Mitomycin-treated T cells are used as stimulator cells in mixed lymphocyte reactions and as feeder cells for growth of B cell colonies, the assumption being that mitomycin prevents proliferation of T cells. We tested this assumption by comparing the proliferation of mitomycin-treated T cells in response to stimulation with phytohemagglutinin (PHA) with that of untreated T cells in liquid cultures and in T cell colony assay. We found that incorporation of tritiated thymidine by cells from 11 healthy individuals pretreated with 25, 50 and 100 micrograms/ml mitomycin C was reduced to 13, 11 and 8%, respectively, of that of untreated cells when stimulated by an optimal concentration of PHA (10 micrograms/ml) in liquid cultures. However, parallel experiments with aliquots of the same cells showed that pretreatment with 25, 50 or 100 micrograms/ml mitomycin C merely reduced T cell colonies to 49, 45 and 45%, respectively, of untreated cells. In five additional experiments mitomycin 200 and 400 micrograms/ml reduced T cell colony numbers to 47 and 60%, respectively. Treatment of T cells with 9000 rad completely abolished T cell colony formation. Lower doses of radiation up to 6000 R did not abolish T cell colony formation, although it effectively blocked T cell proliferation to PHA in liquid cultures. 24 h preincubation of T cells with suboptimal doses of PHA and then treatment with mitomycin or radiation did not abolish T cell colony formation. T cells were recovered from the mitomycin-resistant T cell colonies and stimulated in liquid cultures with PHA, untreated or after exposure to 25 micrograms/ml mitomycin C. Incorporation of tritiated thymidine by the mitomycin-treated cells was reduced to 8% of the untreated controls. Our observations suggests: There may be inaccuracies in B cell colony assays using mitomycin-treated T cells because of significant T cell colony formation. There is a population of T cells in the peripheral blood of normal individuals which form colonies and are resistant to mitomycin.

B-Lymphocytes↗

PubMed2002

7-N-(mercaptoalkyl)mitomycins: implications of cyclization for drug function.

The Kyowa Hakko Kogyo and Bristol-Myers Squibb companies reported that select mitomycin C(7) aminoethylene disulfides displayed improved pharmacological profiles compared with mitomycin C (1). Mechanisms have been advanced for these mitomycins that differ from 1. Central to many of these hypotheses is the intermediate generation of 7-N-(2-mercaptoethyl)mitomycin C (5). Thiol 5 has been neither isolated nor characterized. Two efficient methods were developed for mitomycin (porfiromycin) C(7)-substituted thiols. In the first method, the thiol was produced by a thiol-mediated disulfide exchange process using an activated mixed mitomycin disulfide. In the second route, the thiol was generated by base-mediated cleavage of a porfiromycin C(7)-substituted thiol ester. We selected four thiols, 7-N-(2-mercaptoethyl)mitomycin C (5), 7-N-(2-mercaptoethyl)porfiromycin (12), 7-N-(2-mercapto-2-methylpropyl)mitomycin C (13), and 7-N-(3-mercaptopropyl)porfiromycin (14), for study. Thiols 5 and 12-14 differed in the composition of the alkyl linker that bridged the thiol with the mitomycin (porfiromycin) C(7) amino substituent. Thiol generation was documented by HPLC and spectroscopic studies and by thiol-trapping experiments. The linker affected the structure of the thiol species and the stability of the thiol. We observed that thiols 5 and 12 existed largely as their cyclic isomers. Evidence is presented that cyclization predominantly occurred at the mitomycin C(7) position. Correspondingly, alkyl linker substitution (13) or extension of the linker to three carbons (14) led to enhanced thiol stability and the predominant formation of the free thiol species. The dominant reaction of thiols 5 and 12-14 or their isomers was dimerization, and we found no evidence that thiol formation led to mitosene production and aziridine ring-opening. These findings indicated that thiol generation was not sufficient for mitomycin ring activation. The potential pharmacological advantages of mitomycin C(7) aminoethylene disulfides compared with 1 is discussed in light of the observed thiol cyclization pathway.

Animals↗

PubMed2001

Toxic effects of mitomycin-C on cultured ciliary process cells and trabecular meshwork cells.

Mitomycin-C has recently become an adjunct medication for inhibition of fibroblast proliferation in glaucoma filtering procedures. Prolonged postoperative ocular hypotony has been a frequent complication of trabeculectomy with mitomycin-C. In order to characterize the hypotony mechanism, we compared the toxic effects of mitomycin-C on cultured rabbit ciliary process cells and trabecular meshwork cells. The results indicate that mitomycin-C has a more marked effect on ciliary process cells on 3H-thymidine uptake than on trabecular meshwork cells at concentrations ranging from 10(-1) to 10(-5) mg/ml after 3-, 5- and 60-min treatment, respectively. The living cells after mitomycin-C treatment were estimated with MTT assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of mitomycin-C for 3, 5, and 60 min, the cellular MTT values in ciliary process cells were more decreased than in trabecular meshwork cells. Depolarization of the trabecular meshwork cells with 50 mM KCl led to an increase in intracellular calcium concentration, whereas application of mitomycin-C at 10(-3) mg/ml resulted in decrease of KCl-induced intracellular calcium increase. Mitomycin-C (10(-3) mg/ml) decreased cAMP concentration in ciliary process cells following 3- and 5-min treatment; however, it did not significantly affect the cellular cAMP concentration after only a 1-min exposure. Mitomycin-induced marked ladder pattern of DNA fragmentation was observed in ciliary process tissues after treatment with 10(-1) mg/ml of mitomycin-C for 3 and 5 min. However, the DNA pattern in trabecular meshwork tissues was not obviously affected by mitomycin-C. These findings from our results indicate that mitomycin-induced ocular hypotony may result from damage to both ciliary process and trabecular meshwork tissues.

Animals↗

PubMed1992

Bioactivation of mitomycin C by xanthine dehydrogenase from EMT6 mouse mammary carcinoma tumors.

BACKGROUND: Mitomycin C is an antineoplastic antibiotic requiring bioactivation to an alkylating species or to an intermediate capable of generating oxygen radicals for its toxic effect. The enzymes responsible for the in vivo activation of mitomycin C have been proposed to include NADPH-cytochrome-c reductase, DT-diaphorase, and xanthine oxidase. PURPOSE: In this study, xanthine dehydrogenase, an enzyme structurally similar to xanthine oxidase, was assessed for its ability to activate mitomycin C. Partially purified xanthine dehydrogenase, from EMT6 mouse mammary tumors, was investigated for its ability to bioactivate mitomycin C under both aerobic and hypoxic conditions. METHODS: We conducted this analysis by measuring mitomycin C-induced oxygen consumption, alkylating potential, and mitomycin C consumption and metabolite formation as determined by high-pressure liquid chromatography analysis. RESULTS: Bioactivation of mitomycin C by xanthine dehydrogenase under both aerobic and hypoxic conditions gave rise to the formation of a metabolite, 2,7-diaminomitosene. Formation of this metabolite and alkylating ability were greater under hypoxic than under aerobic conditions and were increased when the pH was decreased from 7.4 to 6.0. Mitomycin C consumption was the same under both aerobic and hypoxic conditions and was independent of pH. Oxygen consumption studies showed that xanthine dehydrogenase-activated mitomycin C consumed oxygen at a much lower rate than xanthine oxidase-activated mitomycin C. CONCLUSIONS: Xanthine dehydrogenase-activated mitomycin C appears to be a good alkylating species but a relatively poor generator of reactive oxygen when compared with xanthine oxidase activation under aerobic conditions. IMPLICATION: Xanthine dehydrogenase may play an important role in the bioactivation of mitomycin C to an alkylating species under both aerobic and hypoxic conditions.

Animals↗

PubMed2006

Chemo-prevention in superficial bladder cancer using mitomycin C: a survey of the practice patterns of British urologists.

OBJECTIVE: To assess the use of mitomycin C, by urologists within the UK, as a single-dose intravesical agent. Current European recommendations are to use one dose after any new tumour resection. METHODS: We assessed the current patterns of mitomycin C usage amongst British urologists, particularly with reference to one instillation after resecting a new bladder tumour, hypothesizing that British urologists would use mitomycin C in line with current guidelines. A one-page questionnaire was mailed to 527 consultant urologists in the UK enquiring about their use of mitomycin C in superficial bladder cancer. A second mailing was sent to encourage nonresponders. RESULTS: Of the 527 consultants, 320 (61%) replied, of which 313 (59%) questionnaires were evaluable. Of these 313 respondents, 299 (95%) used mitomycin C; 244 respondents (82%) advocated the use of one dose of mitomycin C after resecting a new tumour, but only 10 (4%) would use it immediately after tumour resection and 155 (64%) use it within 24 h. Most (98%) respondents favoured the use of a mitomycin C course after resecting multiple tumours or after multiple recurrences. Interestingly, 20 respondents (7%) would use mitomycin C as a first-line therapy for carcinoma in situ and a further 23 (8%) would use it for G3T1 tumours. A minority (14%) would use it after nephrectomy for upper tract transitional cell carcinoma. Almost all respondents indicated a dose of 40 mg in 40 mL of diluent. Maintenance treatment with mitomycin C was advocated by 44 (15%) of respondents, mainly for recurrent multifocal Ta/T1 tumours. The perception of the side-effects of mitomycin C was favourable, with 69% of respondents judging mitomycin C to be well tolerated with mild side-effects. CONCLUSION: Urologists adopt new ideas rapidly, as shown by the wide acceptance of the UK Medical Research Council study. The prompt use of mitomycin C needs to be reinforced, as efficacy is optimum within 6 h of resection. A few consultants persist in continuing with established practices, which have little evidence base. The publication of such survey results, with guidelines for treatment, should encourage those urologists whose practice is at variance from the norm to reflect on and change their practice.

Administration, Intravesical↗

PubMed2000

Trabeculectomy with mitomycin C for post-keratoplasty glaucoma.

AIM: To investigate the effect of trabeculectomy with and without mitomycin C in post-keratoplasty glaucoma. METHODS: A retrospective study was performed on patients who underwent trabeculectomy for glaucoma after penetrating keratoplasty. 34 eyes of 32 patients were included in this study. 26 eyes received trabeculectomy with mitomycin C and eight eyes without mitomycin C. The procedure was deemed successful if the intraocular pressure was maintained below 21 mm Hg with or without use of additional antiglaucoma medication (mean follow up time 22.3 (SD 10.3) months). RESULTS: At the last examination trabeculectomy was successful in 19 of 26 eyes (73.0%) with mitomycin C (+) and two of eight (25.0%) without (p=0.0219). When the prognosis was analysed by Kaplan-Meier curve, the mitomycin C (+) group showed a better prognosis (p=0.0182). Mean intraocular pressure and average number of glaucoma medications improved in the group with mitomycin C without severe side effects on the graft. Graft rejection after trabeculectomy was seen in two eyes in the mitomycin C group. Final graft clarity rate was 69.2% (18/26) in the mitomycin C (+) group and 37.5% (3/8) in the mitomycin C (-) group. Complications such as persistent epithelial defect, cystoid macular oedema, choroidal detachment, leakage from bleb were seen in four eyes in the mitomycin C (+) group and in one eye in the mitomycin C (-) group. CONCLUSIONS: Trabeculectomy with mitomycin C showed better results for glaucoma following keratoplasty.

Adult↗

PubMed1984

Cytotoxicity of mitomycin C on clonogenic human carcinoma cells is not enhanced by hypoxia.

The bioreductive alkylating agent mitomycin C (mitomycin) has been shown to have greater activity under hypoxic than oxic conditions on murine cell lines such as the EMT-6 fibrosarcoma cell line. Solid tumors are known to contain hypoxic cells and are relatively resistant to ionizing radiation and some chemotherapeutic agents. We tested the cytotoxicity of mitomycin against fresh biopsies of human carcinomas under both hypoxic and oxic conditions in the human tumor clonogenic assay (HTCA). Additionally, we examined the metabolism of mitomycin by sonicates of the murine EMT-6 cells and the human WiDR colon carcinoma cells. We confirmed that under our clonogenic assay conditions the EMT-6 cell line was more sensitive to mitomycin under hypoxic than oxic conditions. Additionally, we established that EMT-6 cells also metabolize mitomycin at a more rapid rate under hypoxic than oxic conditions. However, these effects of hypoxia on mitomycin activity were not demonstrable for the human WiDR colon cancer cell line. In addition to these findings, the cytotoxicity of mitomycin was either unchanged or reduced under hypoxic conditions for ten fresh human tumors tested for mitomycin sensitivity in HTCA. Based on these observations, we conclude that the potentiating effect of hypoxia on mitomycin metabolism and biological activity may be peculiar to the murine EMT-6 and S-180 cell lines and that mitomycin C is not likely to have differential efficacy against hypoxic human carcinoma cells.

Animals↗

PubMed1988

Mitomycin C is not metabolized by but is an inhibitor of human kidney NAD(P)H: (quinone-acceptor)oxidoreductase.

It has been suggested that quinone reductase [NAD(P)H: (quinone-acceptor)oxidoreductase], also known as DT-diaphorase, protects hypoxic cells against mitomycin C cytotoxicity by metabolizing mitomycin C to less toxic metabolites. This hypothesis is based on an increase in mitomycin C's cytotoxicity in the presence of the potent quinone reductase inhibitor dicumarol. It has been suggested that under aerobic conditions the metabolism of mitomycin C by quinone reductase leads to the formation of cytotoxic metabolites. In the present study, mitomycin C was found not to be a substrate for partially purified quinone reductase from human kidney. Mitomycin C did not cause the oxidation of NADPH by quinone reductase and there was no utilization of mitomycin C and no appearance of its metabolites. Quinone reductase did not catalyze the formation of alkylating metabolites from mitomycin C, determined by the lack of formation of 4-(p-nitrobenzyl)pyridine conjugates. However, mitomycin C was a weak competitive inhibitor of quinone reductase with dichloroindophenol as the substrate, with Ki = 0.32 mM. Therefore, the alteration of mitomycin C's cytotoxicity by dicumarol in tumor cell lines appears to involve a mechanism other than the direct inhibition of mitomycin C reduction by quinone reductase.

2,6-Dichloroindophenol↗

PubMed1993

The case for mitomycin in non-small cell lung cancer.

The activity of mitomycin in non-small cell lung cancer (NSCLC) has been well documented in both single-institution pilot and multi-institution randomized trials. Despite the inclusion of patients ineligible for current trials of newer single agents due to poor performance status, prior irradiation to indicator lesions, or prior chemotherapy, mitomycin emerges from such prior studies as the most consistently active single agent currently available for NSCLC. While randomized trials in stage IV disease demonstrate an improved response rate with mitomycin and cisplatin in comparison with cisplatin alone (p = 0.03), and with mitomycin, vindesine, and cisplatin in comparison with vindesine and cisplatin alone (p = 0.003), the potential with mitomycin is most apparent with weekly bolus or infused vindesine/vinblastine, and higher-dose cisplatin (MVP) in neoadjuvant approaches to stage III disease. Indeed, four trials of neoadjuvant MVP in predominantly stage IIIA (bulky N2) disease produced a consistent 19-month median and 26-33% 3-year survival, which is to be compared with 8 months and 6%, respectively, with traditional thoracic irradiation alone. Nonetheless, prior use of mitomycin without guidelines for cumulative dose, schedule, or guidelines for use in combined-modality therapy has produced widespread frustration stemming not only from frequent antineoplastic effect, but also from frequent toxicity. The several related syndromes of mitomycin-associated thrombotic microangiopathy, ranging from hemolytic-uremic syndrome to pulmonary injury, appear avoidable through limiting the cumulative mitomycin dose to a maximum of 30 mg/m2; scheduling mitomycin at not less than 4- to 6-week intervals; perioperative use of corticosteroids and low inspired oxygen; and close patient follow-up. While dexamethasone preceding mitomycin reduces the frequency and severity of mitomycin-associated lung injury (p = 0.0005), dexamethasone premedication also reduces the response rate of MVP in NSCLC (p < 0.025).

Aged↗

PubMed1986

DNA alkylation by enzyme-activated mitomycin C.

After anaerobic reductive activation by either NADPH cytochrome P-450 reductase (EC 1.6.2.4) or xanthine oxidase (EC 1.2.3.2), mitomycin C readily alkylated DNA. When the mitomycin C-alkylated DNA is digested by DNase, snake venom phosphodiasterase, and alkaline phosphatase, only partial release of the monofunctionally linked mitomycin C nucleotide adduct occurs. Cross-linked adducts are not released into dinucleotides but resist nuclease digestion and remain in oligonucleotides and insoluble precipitates. Kinetic analyses show that the nuclease-resistant fraction which is indicative of DNA cross-linking by mitomycin C takes place quite readily. This nuclease-resistant fraction is particularly significant when the amount of total bound mitomycin C is less than 15 mumol/mmol of DNA. The cross-linked mitomycin C product accounts for more than half of the total alkylation under all pH conditions tested. Our data suggest that particular DNA sites are available for DNA cross-linking by mitomycin C, and these sites are probably the preferred and immediate alkylating targets. Furthermore, DNA cross-links by mitomycin C are not the secondary product of monofunctional adducts. Activity of both flavoenzymes is pH dependent, hence, mitomycin C activation and the rate of DNA alkylation are pH dependent. At elevated mitomycin C alkylation of DNA, the highest amount of cross-linking occurs at neutral pH. High pressure liquid chromatographic separation of the nuclease-digested DNA detected one major and two less prominent mitomycin C adducts. These were verified to be mononucleotide mitosene types by UV spectra showing maximum absorbance at 312 and 250 nm. The major adduct was purified and identified as O6-(2'-deoxyguanosyl)-2,7-diaminomitosene by NMR, indicating that the O6 position of guanine is a preferred site in DNA for at least monofunctional linkage formation.

Alkylating Agents↗

PubMed1997

Effect of mitomycin-C on human retinal pigment epithelium in culture.

PURPOSE: To determine the effect of mitomycin-C on confluent and non-confluent human retinal pigment epithelium (RPE) in tissue culture. METHODS: The effect of mitomycin-C on confluent RPE was determined by treating first passage confluent cells with 0.01, 0.1, 1, 10, 100 or 1000 micromolar (microM) mitomycin-C for 1, 3, or 7 days. The cell viability after treatment was determined by using an esterase stain. The effect of mitomycin-C on proliferating RPE was determined by incubating non-confluent cells with the above concentrations of mitomycin-C for 20 min, 1 hour or 24 hours. RESULTS: Mitomycin-C can be toxic to a confluent RPE monolayer, and the LD50 is 421, 28.8 or 0.0632 microM when cells are continually exposed to mitomycin-C for 1, 3 or 7 days, respectively. Exposure to mitomycin-C at concentrations > or = 10 microM for 20-60 min significantly inhibits proliferation of non-confluent RPE. A 24 hour exposure of RPE to 1 microM mitomycin-C markedly inhibits proliferation of non-confluent RPE with minimal toxicity to confluent RPE. CONCLUSIONS: Since exposure of human RPE to mitomycin-C for 24 hours can inhibit cell proliferation at concentrations which are well-tolerated by confluent RPE, mitomycin-C may be a suitable agent for inhibiting RPE proliferation in vivo.

Cell Division↗

PubMed1999

Can topical mitomycin prevent laryngotracheal stenosis?

OBJECTIVES/HYPOTHESIS: Early topical application of mitomycin to a laryngotracheal lesion may prevent or reduce laryngotracheal stenosis (LTS). STUDY DESIGN: Prospective controlled animal study. METHODS: LTS was induced in 60 dogs randomly assigned to four groups. Controls received an immediate topical application of normal saline. The suction-control group received an immediate application of normal saline followed by suction of secretions on day 2. The mitomycin group received immediate application of 0.7 mL mitomycin (0.2 mg/mL). The repeat-mitomycin group received an immediate application of mitomycin and a second application on day 2, after secretions were suctioned. The laryngeal lumens were measured endoscopically at baseline, day 12, and day 21. Animals were euthanatized if stenosis approximated 95% or at day 21. RESULTS: All dogs in the mitomycin groups survived to day 21, compared with 12 in the suction group and only 2 controls. No side effects of mitomycin were observed. At day 21, surviving controls had 85% and 95% stenosis. In the mitomycin group, median stenosis was 27% (interquartile range, 29% to 42%); in the repeat-mitomycin group, 30% (22% to 40%); and in the suction-control group, 84.5% (72.5% to 93.5%). The mitomycin group differed significantly from controls on day 12 (median difference = 85%, 95% CI = 80%-94%, P < .0001) and day 21 (difference = 63.9%, 95% CI = 58%-85%, P = .031). CONCLUSION: A single topical application of mitomycin significantly reduces the severity of LTS in dogs. Reapplication after 2 days does not improve results. Prospective clinical studies are warranted to assess the efficacy in humans.

Administration, Topical↗

PubMed1992

Bioreductive activation of mitomycin C by DT-diaphorase.

The role of DT-diaphorase (DTD, EC 1.6.99.2) in the bioreductive activation of mitomycin C was examined using purified rat hepatic DTD. The formation of adducts with reduced glutathione (GSH), binding of [3H]mitomycin C to DNA, and mitomycin C-induced DNA interstrand cross-linking were used as indicators of bioactivation. Mitomycin C was metabolized by DTD in a pH-dependent manner with increasing amounts of metabolism observed as the pH was decreased from 7.8 to 5.8. The major metabolite observed during DTD-mediated reduction of mitomycin C was 2,7-diaminomitosene. GSH adduct formation, binding of [3H]mitomycin C and mitomycin C-induced DNA interstrand cross-linking were observed during DTD-mediated metabolism. In agreement with the pH dependence of metabolism, increased bioactivation was observed at lower pH values. Temporal studies and experiments using authentic material showed that 2,7-diaminomitosene could be further metabolized by DTD resulting in the formation of mitosene adducts with GSH. DNA cross-linking during either chemical (sodium borohydride) or enzymatic (DTD) mediated reduction of mitomycin C could be observed at pH 7.4, but it increased as the pH was decreased to 5.8, showing the critical role of pH in the cross-linking process. These data provide unequivocal evidence that the obligate two-electron reductase DTD can bioactivate mitomycin C to reactive species which can form adducts with GSH and DNA and induce DNA cross-linking. The use of mitomycin C may be a viable approach to the therapy of tumors high in DTD activity, particularly when combined with strategies to lower tumor pH.

Animals↗

PubMed1986

Cytotoxicity and DNA lesions produced by mitomycin C and porfiromycin in hypoxic and aerobic EMT6 and Chinese hamster ovary cells.

Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under conditions of oxygen deficiency; however, mitomycin C produced considerably more DNA cross-linking than did porfiromycin in oxygenated cells. DNA interstrand cross-links were observed in hypoxic EMT6 cells throughout a 24-h period following removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C and porfiromycin at 6 h after drug removal, with a decrease in these lesions being observed by 24 h, suggesting that the rate of formation of the cross-links may be slower and the removal of cross-links more rapid under aerobic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Mitomycin and the human corneal endothelium.

OBJECTIVE: To investigate the ultrastructural and physiologic effects of exposure of the human corneal endothelium to mitomycin at concentrations of 20 micrograms/mL and 200 micrograms/mL using electron microscopy and in vitro specular perfusion techniques. METHODS: Four pairs of corneas (with one cornea of each pair receiving balanced salt solution [BSS Plus, Alcon Laboratories, Fort Worth, Tex] and the other receiving BSS Plus with 20 micrograms/mL of mitomycin) suitable for transplantation, except for extremes of age or systemic disease, underwent perfusion with corneal thickness measured serially every 15 minutes followed by fixation for electron microscopy. Mean corneal swelling rate was calculated for all four experiments, and the control group that received BSS Plus was compared with the group that received mitomycin using a paired t test. Electron micrographs were examined in a masked fashion. Similar studies were performed using two pairs of corneas that received 200 micrograms/mL of mitomycin. RESULTS: The mean swelling rate for corneas perfused with 20 micrograms/mL of mitomycin (-4.1 microns/h) was not significantly different from that seen in tissue perfused with BSS Plus (-4.2 microns/h). No consistent ultrastructural changes could be attributed to exposure to 20 micrograms/mL of mitomycin. Perfusions of mitomycin at 200 micrograms/mL resulted in prompt corneal swelling with marked ultrastructural alterations compared with tissue perfused with BSS Plus. CONCLUSION: Human corneal endothelium may be exposed to undiluted (200 to 500 micrograms/mL) mitomycin with inadvertent entry into the anterior chamber during dissection of the scleral flap bed in trabeculectomy followed by application of mitomycin. This will result in prompt destruction of the endothelium. Exposure to 20 micrograms/mL of mitomycin, a level exceeding the concentration that may be present in the aqueous humor after its proper application, appears nontoxic in this system.

Endothelium, Corneal↗